The applicant's long-term goal is to understand molecular mechanisms of cardiac cell differentiation. The zebrafish will be used as a model system to study heart formation. In all vertebrates, the early heart tube is composed of only two cell types, the outer myocardial and inner endocardial cells. In this proposal the applicant aims to investigate the origin and differentiation of the endocardial cells and their role in heart formation. Through large scale mutagenesis screens, the applicant has already identified several mutations that specifically affect the formation of the early heart tube; one in particular, cloche, selectively deletes the endocardial cells. In cloche mutants, the myocardial tube forms in the absence of the endocardium but is dysmorphic and exhibits a reduced contraction. The proposed aims are: (1) to determine the migration pathways and analyze the lineage of endocardial cells; (2) to further characterize the cloche mutation, identify additional cloche alleles, map the cloche mutation genetically, isolate the cloche gene by positional cloning, and identify other components in the pathway of endocardial cell differentiation; and (3) to determine the role of endocardial cells in myocardial cell maturation and function.